Metformin prevents stroke damage in non-diabetic female mice with chronic kidney disease

Chronic kidney disease (CKD) worsens ischemic stroke severity in both patients and animals. In mice, these poorer functional outcomes are associated with decreased brain activity of AMP-activated protein kinase (AMPK), a molecule that recently emerged as a potential therapeutic target for ischemic stroke. The antidiabetic drug metformin, a well-known activator of AMPK, has improved stroke outcomes in diabetic patients with normal renal function. We investigated whether chronic metformin pre-conditioning can rescue AMPK activity and prevent stroke damage in non-diabetic mice with CKD. Eight-week-old female C57BL/6J mice were assigned to CKD or SHAM groups. CKD was induced through right kidney cortical electrocautery, followed by left total nephrectomy. Mice were then allocated to receive metformin (200 mg/kg/day) or vehicle for 5 weeks until stroke induction by transient middle cerebral artery occlusion (tMCAO). The infarct volumes were lower in CKD mice exposed to metformin than in vehicle-treated CKD mice 24 h after tMCAO. Metformin pre-conditioning of CKD mice improved their neurological score, grip strength, and prehensile abilities. It also enhanced AMPK activation, reduced apoptosis, increased neuron survival and decreased microglia/macrophage M1 signature gene expression as well as CKD-induced activation of the canonical NF-κB pathway in the ischemic lesions of CKD mice.

Statistical analysis was performed using a non-parametric Spearman correlation test.

Transient middle cerebral artery occlusion (tMCAO)
Ischemic lesions were induced in both SHAM and CKD mice 5 weeks after the last SHAM or CKD surgery (i.e. in 15-week-old mice). Briefly, mice were anesthetized with ketamine (80 mg/kg) plus xylazine (8 mg/kg) and a 20-mm-long 6-0 silicon rubber-coated nylon monofilament (Doccol®, Sharon, Massachusetts, USA) was inserted into the right common carotid artery. The diameter of the coated filaments was 0.21 ± 0.02 mm (reference 6021910PK5Re). MCAO sutures in this category of coated tip diameter are suitable for MCAO in animals with a body weight in the range of 22 ± 2 g. The filament was then advanced to the internal carotid artery and passed into the intracranial circulation (12-13 mm distal to the carotid bifurcation), thus occluding the origin of the MCA. The right MCA was occluded for 15 min. The filament was then carefully removed to produce the reperfusion. Animals were allowed to recover for 24 h and then euthanized for infarct volume analysis.

Neurological evaluation
-Neuroscore: A six-grade neuroscore was used to assess post-ischemic motor and behavioral impairments. Mice were graded 0 to 5 as follows. Grade 5: mice were held gently by the tail one meter above the ground and observed for forelimb flexion. Normal mice extended both forelimbs toward the floor. Mice that extended both forelimbs toward the floor and did not display other neurological impairment were assigned a grade of 5. Grade 4: mice with consistent flexion of the forelimb contralateral to the injured hemisphere (varying from mild wrist flexion and shoulder adduction to severe posturing, with full flexion of the wrist and elbow, and induction of the shoulder with internal rotation) were assigned a grade of 4. Grade 3: mice were placed on a large sheet of soft, plastic-coated paper that they could grip firmly with their claws. The experimenter held the mouse by the tail and applied gentle lateral pressure to the animal's shoulder until the forelimbs slid several centimeters. The maneuver was repeated several times to the left and to the right. Normal mice and slightly impaired mice resisted sliding to an equivalent extent in each direction. However, severely impaired mice with consistently reduced resistance to pushing towards the paretic side were assigned a grade of 3. Grade 2: mice were then allowed to move about freely and observed for circling behavior when their tail was pulled. Mice that circled consistently towards the paretic side were assigned a grade of 2.
Grade 1: mice were allowed to move about freely and were observed for circling behavior.
Mice that circled spontaneously and consistently toward the paretic side were assigned a grade of 1. Grade 0: mice without any spontaneous motion were assigned a grade of 0.
-Prehensile test: A prehensile test was performed using a horizontal stainless-steel wire (length: 60 cm, diameter: 3 mm) placed 40 cm above a foam pad. The wire was graded into 6 equal parts starting from the point of suspension till the platform on each side. The forepaws of the mice were placed on the wire in the centre and the animals were left suspended for 20 seconds.
The time until the mice fell, their ability to grab the wire with a hind paw, and their motor coordination were measured. The tested animals were scored as follows: 1, 2, 3 or 4 points for holding onto the wire for less than 5 seconds, 6 to10 seconds, 11 to 15 seconds or more than 15 seconds respectively. Mice were scored 0 if they fell immediately after being suspended. An additional point was added if the mice managed to grab the wire with a hind paw. If they managed to advance to either side, mice were given 1, 2, 3, 4 or 5 points for reaching grading 1, 2, 3, 4 and 5 respectively. An extra point was granted if they were able to reach the platform.
-Grip-test: The muscular strength of the forelimbs was assessed using a grip strength test (Bioseb, Vitrolles, France). A grip strength meter was positioned horizontally and the mice were held by the tail and lowered towards the apparatus. The animals were allowed to grab the metal grid and were then pulled backwards in the horizontal plane. The force applied to the grid just before the animals lost grip was recorded as the peak tension. The muscular strength of the forelimbs was assessed three times per session and the mean of the three measurements was used for evaluation.

Immunohistochemical examination of the ischemic area
The sections used for immunohistology were cut to a thickness of 20 µm, i.e. thinner than sections used for cresyl violet staining, to facilitate antibody penetration into the brain tissue.
Sections were fixed with 4% ice-cold PFA for 5 min at room temperature (RT) and incubated in sodium citrate (1M, pH = 6) for 20 min at 100°C for antigen retrieval. Sections were then